Vitamin C (ascorbate) is essential in eukaryotic cells as an antioxidant and enzyme cofactor. It protects against cancer and aging by preventing DNA oxidation. Ascorbate is intimately involved in cellular redox homeostasis through its interactions with NADH, glutathione and vitamin E metabolisms. In spite of its importance, there remain significant gaps in our understanding of ascorbate metabolism and regulation. A class of membrane proteins, cytochrome b561s, mediating ascorbate-dependent transmembrane electron transfer, has recently been identified in mammals and plants. Little is known about their biochemical properties or precise physiological functions. In mammals cytochrome b561 isozymes are involved in iron uptake and neuropeptide hormone biosynthesis. In humans and mice the cytochrome b561 protein family includes four proteins. Because of the implications to human biology, Mus musculus is an ideal model system to thoroughly characterize members of this family of redox proteins. Biochemical, molecular and cell biological approaches will be integrated to provide a detailed understanding of the physiological functions and properties of two cytochrome isozymes, likely to mediate important and diverse electron transfer reactions, by addressing the following questions: 1) What are the biochemical and physicochemical properties of these cytochrome b561s? We will characterize the biochemical and physicochemical properties of recombinant mouse cytochrome 13561isozymes and test the hypothesis that these proteins transport electrons across membranes using ascorbate as an electron donor. 2) What are the tissue expression patterns and subcellular distribution of cytochrome b561s? The isozyme-specific tissue expression patterns and subcellular Iocalizations of the cytochrome b561s reflect their physiological functions. We will examine the tissue and subcellular distribution of two cytochrome b561s from mice. 3) What proteins interact with cytochrome b561s? The working hypothesis is that cytochrome b561 isozymes interact with other proteins. These interactions will be explored by using the yeast two-hybrid system. The proposed studies will establish the groundwork for understanding the physiological roles of cytochromes b561. Based on the importance of ascorbate to mammalian physiology, these studies will provide fundamentally new insights into redox metabolism in eukaryotic cells.